Identification of 2S albumin from pomegranate seeds as an IgE-binding protein

Background:The consumption of pomegranate is increasing as itis considered a human health-promoting food. However, it can trig-ger mild to severe allergic reactions. So far several allergens havebeen identified in its red pulp, including Pun g 1 (LTP), Pun g 7 (pea-maclein like-protein) and Pun g 14 (chitinase), whereas any possibleallergens of the pomegranate seeds are still unknown. Objectives.The aim of this study was the investigation of possible allergens con-tained in pomegranate seeds. Method:2S albumin was isolated from the fruit seeds, analyzed bydirect protein sequencing and identified by mass spectrometry. Tenpatients allergic to pomegranate were analyzed by IgE-immunoblot-ting and 4537 randomly-selected subjects were tested for IgE bind-ing to a pomegranate seed extract by the FABER®test. Results:Of these 4537 patients analyzed by FABER®test, 266(6%) proved to be sensitized to at least one of the 5 pomegranateallergenic preparations, Pun g [Seed], Pun g 1, Pun g 14, Pun g 7and Pun g 5 with a prevalence of 54%, 54%, 23%, 13% and 6%, respectively. Ten subjects allergic to pomegranate and analyzed byimmunoblotting showed IgE recognition of a main protein compo-nent of the seed extract. Therefore, this IgE-binding molecule waspurified by chromatographic separations and characterized. Directprotein sequencing revealed a blocked N-terminal residue. Massspectrometry analysis showed a molecular mass of 16 496 Da andpeptidemassfingerprinting allowed its identification as the proteinreported in the Uniprot database with the accession numberA0A218XU94 and classified as uncharacterized protein. The homol-ogy search performed in the Allergome database displayed a similar-ity with 2S albumin storage proteins, such as the allergens Cor a 14,Jug n 1 and Pis v 1. In line with the well-known low degree of aminoacid sequence conservation among 2S albumins, the overall identityobserved between the pomegranate protein (Pun g 2S albumin) andother homologs is quite low, whereas a high identity is limited tospecific protein regions. Conclusion:Pomegranate seed is a sensitizing source. The mainprotein component, Pun g 2S albumin, is a newly identified IgE-bind-ing protein of about 16.5 kDa. Its future inclusion in the FABER®multiplex test will allow us to establish if it represents the main aller-gen in pomegranate seed and the relationships with other seed-derived 2S albumins, thus improving the allergy diagnosis to plant-derived foods.